| An in vitro Transcription/translation System for Detection of Protein Interaction
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Author:
Date:
2016-05-05
[Abstract] Studying protein-protein interaction is crucial to understand the fundamental processes of molecular biology. High-throughput screening, such as immunoprecipitation followed by proteomic analysis, allows for the identification of numerous candidate partners that might interact with a selected protein. However, experimental validation of protein-protein interaction requires conventional cloning and recombinant protein expression/purification, which are complicated and labor-intensive techniques. Here, we demonstrate an efficient experimental pipeline for verifying protein-protein interactions between a bait protein using the example of Odontoglossum ringspot virus (ORSV) capsid protein (CP) and the host CP-binding protein. These candidate CP-binding proteins were identified ...
[摘要] 研究蛋白质 - 蛋白质相互作用对于理解分子生物学的基本过程是至关重要的。高通量筛选，如免疫沉淀，然后蛋白质组分析，允许鉴定可能与选择的蛋白质相互作用的许多候选伙伴。然而，蛋白质 - 蛋白质相互作用的实验验证需要常规克隆和重组蛋白表达/纯化，这是复杂和劳动密集型技术。在这里，我们演示了一个有效的实验管道，用于验证使用Odontoglossum环斑病毒（ORSV）衣壳蛋白（CP）和宿主CP结合蛋白的例子的诱饵蛋白之间的蛋白质 - 蛋白质相互作用。这些候选CP结合蛋白通过高通量蛋白质组学和转录组学方法进行鉴定。使用TOPO克隆策略，将每个候选基因克隆到表达载体中，用于在体外转录/翻译系统的单个步骤中表达His标记的重组蛋白。这种表达的His标记的候选物可以在共免疫沉淀（co-IP）测定中用作CP诱饵蛋白的猎物，以验证它们的物理相互作用。不需要传统的蛋白质表达和纯化，该管道简化了验证过程，并为高通量蛋白质 - 蛋白质相互作用研究提供了解决方案。
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| Northwestern Blot of Protein-RNA Interaction from Young Rice Panicles
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Author:
Date:
2013-04-05
[Abstract] The northwestern assay is employed to study the interaction between protein and RNA. The RNA binding proteins tend to bind to different kinds of RNA through either known domains or unknown sequences of proteins. Rice LGD1 recombinant protein, a grass-specific novel protein with RNA binding sequences in its C-terminal, was used to probe its function as an RNA binding protein. The LGD1 comprising von Willebrand factor type-A domain (vWA), coiled-coil and nuclear localization signal (NLS) is a class of protein that localizes both in the nucleus and cytoplasm. Although LGD1 does not contain any putative RNA binding domains, we could find high-affinity RNA binding residues at the C-terminus using ‘RNABindR’ prediction software (Terribilini et al., 2007). The LGD1 recombinant protein, ...
[摘要] 使用西北测定来研究蛋白质和RNA之间的相互作用。 RNA结合蛋白倾向于通过蛋白质的已知结构域或未知序列结合不同种类的RNA。将水稻LGD1重组蛋白（一种在其C末端具有RNA结合序列的草特异性新蛋白）用于探测其作为RNA结合蛋白的功能。包含von Willebrand因子A型结构域（vWA），卷曲螺旋和核定位信号（NLS）的LGD1是一类定位于细胞核和细胞质中的蛋白质。虽然LGD1不包含任何推定的RNA结合结构域，我们可以使用'RNABindR'预测软件在C末端找到高亲和力RNA结合残基（Terribilini等人，2007）。从细菌纯化的LGD1重组蛋白以某种方式甚至在变性条件下形成二聚体和单体。然而，仅二聚体形式能够结合总RNA和mRNA。由于其重现性和可靠性，我们认为该协议可以用于不同的生物。
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